1) Field of the Invention
The present invention relates to an apparatus for measuring a medical substance where a resonance phenomenon with an evanescent wave, such as a surface plasmon resonance phenomenon, is used; a sensor for use in the apparatus; and a sensing element for use in the sensor. Particularly, the present invention can be preferably applied for measuring medical substances contained in body fluids, such as urine or blood, for example, organic medical substances, such as morphine, methamphetamine, cocaine and diacetylmorphine, which act as an antigen having a small molecular weight.
2) Prior Art
Many methods for detecting medical substances contained in body fluids such as urine or blood have been suggested. For instance, chromatography analysis, mass spectroscopy analysis, precipitation reaction analysis and spectrochemical analysis can be mentioned. However, according to these conventional methods, complicated pretreatment process, for instance, extracting or refining, is required in order to adjust a sample liquid for use in detection. Furthermore, the analyzing sensitivity of these conventional methods is so low as not to be able to use practically.
On the other hand, nowadays, attention is being made to a new method for analyzing medical substances contained in body fluid where a resonance phenomenon with an evanescent wave is used, because according to the new method it can be expected to measure medical substances with a high level of sensitivity. The evanescent wave is a light wave which is exponentially attenuated in accordance with the distance from a boundary so that the light wave substantially has no energy, for example, like a light wave which is generated when light is totally reflected.
A well known resonance phenomenon with an evanescent wave is a surface plasmon resonance phenomenon which is generated when the wave number of evanescent waves becomes coincident with the wave number of the surface plasmon. When the surface plasmon resonance phenomenon is generated, a part of the energy of light is used to excite the surface plasmon, so that the generation of the surface plasmon resonance can be obtained as a reduction of the energy of the light. Since the condition for generating the surface plasmon resonance phenomenon, i.e. an incident angle or a wave number of the incident light, varies according to the condition of the substance which is made to contact with the sensing layer, information about the condition of the substance can be obtained by examining the condition for generating the surface plasmon resonance phenomenon.
For instance, when a thin metal film is formed on a surface of a prism and a light is made incident upon the prism to be reflected by the boundary surface of the prism and the thin metal film, the evanescent wave goes out of the prism and then propagates along the surface of the prism. If the metal film is sufficiently thin, the evanescent wave passes through the metal film. The wave number of the evanescent wave depends on the incident angle of the incident light. On the other hand, the surface plasmon is an elementary excitation of a surface plasma oscillation, which corresponds to the fact that the quantum, i.e. an energy, of the plasma oscillation localized on the surface is quantized. A metal can be considered as a solid-state plasma where free electrons are moving in the background of the fixed cations, and therefore a surface plasmon can be generated on the surface of the metal. The wave number of the surface plasmon depends upon a refractive index of the substance being made to contact with the thin metal film.
When the refractive index is varied by the fact that the condition of the substance being made to contact with the thin metal film is changed, the wave number of the surface plasmon is varied and then the wave number of the evanescent wave is also varied. That is to say, the wave number of the evanescent wave, at which the light intensity of the reflection light decreases, is varied. Since the wave number of the evanescent wave depends on the incident angle of the incident light, the incident angle, at which the light intensity of the reflection light decreases, i.e. a resonance angle, is changed. Therefore, the condition of the substance which is made to contact with the thin metal film, more concretely the refractive index of the substance, can be obtained by reading out the change of the incident angle, i.e. the resonance angle. It should be noted that the greater the refractive index of the substance being made to contact with the thin metal film, the larger the resonance angle, and generally, the heavier the molecular weight of the substance, the greater the refractive index.
A resonance phenomenon using a resonant mirror is also well known as another resonance phenomenon with an evanescent wave. The resonant mirror has a constitution such that a dielectric resonant layer, e.g. a layer made of Titania, having a thickness of 100 nm and a high refractive index is made to contact with a prism via a layer, i.e. a layer made of Silica, having a thickness of 1 μm and a low refractive index. The resonant mirror works in such a manner that when the layer having a low refractive index is sufficiently thin, a light (an evanescent wave) approaches to the dielectric layer having a high refractive index and then a resonance phenomenon is caused. When the wave number of the resonance mode of the layer having a high refractive index is coincident with the wave number of the incident light being made incident upon the prism with a certain incidence angle, the light may couple into the resonant layer efficiently, and then an evanescent light is generated on the sensing interface. The evanescent light can be detected as a change of phase which is caused in accordance with the change of the condition of the substance (more concretely, the change of the refractive index of the substance) being made to contact with the sensing interface. That is to say, when the resonant mirror is used, the refractive index of the substance which is made to contact with the layer having a high refractive index can be known by detecting the incident angle of the incident light at which the phase of the reflected light of the incident light is varied.
In this manner, the resonance phenomenon with the evanescent wave is caused depending upon the refractive index of the substance which is made to contact with the sensing interface. Then, it is tried to preliminarily fix an antibody to the resonance material, i.e. the thin metal film, and then to measure a medical substance which acts as an antigen coupled with the antibody fixed to the material in a specific manner. That is to say, when a sample liquid containing a medical substance is made to contact with the thin metal film, etc. on which the antibody is fixed, the medical substance contained in the sample is coupled with the antibody and then the refractive index is varied. Therefore, the amount of the medical substance contained in the sample liquid can be obtained by comparing the condition (the resonance angle, etc.), when the antibody is fixed to the thin metal film but the medical substance is not coupled with the antibody yet, with the condition (the resonance angle, etc.) after the medical substance is coupled with the antibody.
However, the medical substance contained in a body fluid, such as urine or blood, generally has only some hundreds molecular weight, which is quite small. Therefore, even if such a small molecular weight of substance is coupled with the antibody, a sufficient change of the refractive index cannot be obtained. It means the amount of change of the resonance angle is also very small. Thus, it is difficult to measure such an organic medical substance having a low molecular weight by using the surface plasmon resonance phenomenon in a direct manner.
In order to overcome this problem, a new method is suggested where a standard substance is prepared having a high molecular weight, such as protein, and is coupled with a medical substance to be detected in order to increase the molecular weight, and then the standard substance and the medical substance contained in the sample liquid to be measured are coupled with the antibody by competitive assay to improve the sensitivity. (Japan Chemical Association, 70th annually meeting in spring, “Measurement of methamphetamine using an immunological reaction”, by Niimoto, et al).